1. Field of the Invention
The present invention generally relates to a family of allosteric effectors of hemoglobin and more specifically to chirality affects of allosteric effectors where the chiral carbon has a substituted carbon ring, a heteroatom ring, or different substituents. The invention includes several new potent enantiomers that are superior than their racemic mixture and other enantiomeric isomer, possessing different degrees of allosteric potency.
2. Background Description
Human hemoglobin (Hb) is a tetrameric allosteric protein comprised of two alpha and two beta chains and functions to deliver oxygen from the lungs to the many tissues of the body. The four subunits are arranged around a molecular two fold axis creating a central water cavity. As an allosteric protein, Hb exists in an equilibrium between two states, the relaxed (R) or oxy-state and the tense (T) or deoxy-state. In the oxy-state, the water cavity is narrow and the subunits have fewer and weaker bonds between them (i.e., relaxed). However, in the deoxy-state, the water cavity is larger, and the subunits are tightly tethered together by salt bridges (i.e., tense). The allosteric equilibrium can be influenced by allosteric modifiers. Such molecules can increase the oxygen affinity of Hb shifting the allosteric equilibrium toward oxy-Hb or decrease the affinity of oxygen, shifting the equilibrium to the deoxy-Hb. Modifiers that decrease the oxygen affinity act by adding constraints to the T-state. Oxygen affinity decreasing agents have several potential applications including radiosensitization of tumors, enhancement of oxygen delivery to hypoxic and ischemic tissues, and shelf-life prolongation of stored blood.
The gap between the xcex2 subunits is wide enough for 2,3-diphosphoglycerate (2,3-DPG), a naturally occuring allosteric modifier, to dock in and bind, forming additional salt bridges that further stabilize the deoxy state. Therefore, compounds that lower the affinity of oxygen for Hb do so by strengthening the existing salt bridges or by adding new ones to the tense state.
Several synthetic agents have been reported to lower the affinity of oxygen for Hb. In the search for an antisickling agent, Abraham and coworkers discovered the antilipidemic drug, clofibric acid, that lowered the oxygen affinity of Hb. Perutz and Poyart followed with a report that bezafibrate, another antilipidemic agent, was also a right-shifting compound, more potent than DPG and clofibric acid. Lalezari and coworkers demonstrated that shortening the four atom bridge to a three atom urea bridge produced even more potent allosteric modifiers, but their potential as clinical agents was limited due to loss of activity in the presence of serum albumin.
It has been proposed that influencing the allosteric equilibrium of hemogobin is a viable avenue of attack for treating diseases. The conversion of hemoglobin to a low affinity state is believed to have general utility in a variety of disease states where tissues suffer from low oxygen tension, such as ischemia and radio sensitization of tumors. Several synthetic compounds have been identified which have utility in the allosteric regulation of hemoglobin and other proteins.
It is therefore an object of the present invention to provide a family of compounds which allosterically modifies hemoglobin such that hemoglobin is present in blood in a lower oxygen affinity state.
It is therefore an object of the present invention to provide synthetic agents that can enhance the oxygenation of tissues. Enhancement of oxygenation has several potential therapeutic applications: (1) radio-sensitization of tumors, (2) treatment of stroke and cerebral traumas, (3) shelf-life prolongation of stored blood, (4) treatment of angina and myocardial infarcation, and (5) reduction of surgical blood loss and blood transfusions.
Currently, two of the most potent oxygen-affinity decreasing agents developed by Abraham et al. are shown as RSR13 and JP7 in Table I below. The high resolution crystal structure of the RSR13-Hb complex has been determined. The small molecule binds near the top of the xcex1 subunits and points down the central water cavity to the xcex1,xcex2-subunit interfaces making several important interactions with the protein. RSR46, KDD86, and RSR4 shown in Table I are also oxygen affinity decreasing agents.
Specifically, compounds having substituted chiral centers and the structures: 
wherein R1 and R2 are selected from the group comprising CH3, Cl, and 5 carbon cyclics; R3 is selected from the group comprising H, OH, and OC2H5; R4 and R5 are selected from the group comprising CH3, cyclics containing CH3 substituents, OCH3, C2H5, phenyl and substituted phenyl; and wherein R4 and R5 are not the same, and 
wherein R1 is selected from the group comprising H, CH3, CH(CH3)2, CH2Ph, CH2CH(CH3)2, CH(CH3)C2H5, CH2CH2COOH, CH2COOH, CH2tryptophan, CH2 Indole, CH2PhOH, CH2OH, CH2SCH3, (Me)2SMe, (CH2)3, CH2SCH2Ph, CH(OH)CH3, (CH2)4NHOCOCH2Ph, and (CH2)4NH2. 
Where R3 is selected from the group comprising H, CH3, CH(CH3)2, CH2Ph, CH2CH(CH3)2, CH(CH3)C2H5, CH2CH2COOH, CH2COOH, CH2tryptophan, CH2 Indole, CH2PhOH, CH2OH, CH2SCH3, (Me)2SMe, (CH2)3, CH2SCH2Ph, CH(OH)CH3, (CH2)4NHOCOCH2Ph, (CH2)4NH2 etc. have been identified as being allosteric effectors of hemoglobin.
Investigation of the effect of stereochemistry on activity and binding conformation shows that the existence of a chiral center affects the allosteric activity. Specifically, a chiral center was introduced in compounds having the general structures of RSR13, JP7, RSR4, RSR46 and KDD86 (shown in Table I). The new chiral molecules (class B) were prepared by replacing either one of the gem dimethyl groups of Table I compounds with other alkyl/alkanoic, un/substituted cycloalkyl/cycloalkanoic, substituted aromaatic groups or by condensing the carboxylate group of the parent.molecule (Table 1 compounds) with various D and L isomers of amino acids such as alanine, valine, asparagine, cysteine, glutamic acid, phenylalanine, glycine, histidine, leucine, isoleucine, proline, arginine, serine, threonine, tryptophan, tyrosine, and lysine (class C).
The synthesis of the compounds (class B) involves central intermediate amidophenols: 4-[[(3,5-dimethylanilino)carbonyl]methyl]phenol, 4-[[(5-indanyl)carbonyl]methyl]phenol, and 4-[[(3-chloro-5-methylanilino)carbonyl]methyl]phenol, where 3,5 dimethylaniline or 5-aminoindan is condensed with 4-hydroxyphenylacetic acid in refluxing xylene over a three-day period. While 3,5-dimethylaniline and 5-aminoindan were both readily available. 
The scheme 1 above as well the schemes 2 and 3 shown below were utilized to produce the corresponding racemates. 
The previously reported xcex1-aryloxyisobutyric acid analogs were obtained via reaction of amidophenols with acetone-chloroform in the presence of sodium hydroxide. In this process, the appropriate ketone is substituted for acetone in tetrahydrofuran to obtain the proposed compounds 1-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-3-methylcyclopentane carboxylic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy-2-methylbutanoic acid 1-4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylcyclopentane carboxylic acid, 4-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]tetrahydro-2H-4-pyrancarboxylic acid, 3-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methyltretrahydro-3-furan carboxylic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-3-methoxy-2-methylpropanoic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpentanoic acid, 1-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-3-methylcyclohexane carboxylic acid, 1-[4-[[(5-indanyl)carbonyl]methyl]phenoxy]-3-methyl cyclopentane carboxylic acid, 2-[4-[[(5-indanyl)carbonyl]methyl]phenoxy]-2-methylbutanoic acid, 2-[4-[[(5-indanyl)carbonyl]methyl]phenoxy]-2-methylcyclopentane carboxylic acid, 1-[4-[[(3-chloro-5-methylanalino)carbonyl]methyl]phenoxy]-3-methylcyclopentane carboxylic acid, 2-[4-[[(3-chloro-5-methylanalino)carbonyl]methyl]phenoxy]-2-butanoic acid, 1-[4-[[(3-chloro-5-methylanalino)carbonyl]methyl]phenoxy]-2-methylcyclopentane carboxylic acid.
The schemes 4 and 5 were used to prepare racemates of the compounds 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-proprionic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-fluoroacetic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-butanoic acid, 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-pentanoic acid, 2-[4-[[(3,5-dimethylanilino)cardonyl]methyl]phenoxy]-hexanoic acid, 2-[4-[[(3-chloro-5-methylanilino)carbonyl]methyl]phenoxy]-propionic acid, 2-[4-[[(3-chloro-5-methylanilino)carbonyl]methyl]phenoxy]-butanoic acid, 2-[4-[[(5-indanyl)carbonyl]methyl]phenoxy]-propionic acid, and 2-[4-[[(5-indanyl)carbonyl]methyl]phenoxy]-butanoic acid. 
This method employs condensation of the corresponding amidophenol with the xcex1-bromo ester in the presence of base followed by base hydrolysis of the ester to give the desired xcex1-aryloxy acid product.
Abraham et al. designed and synthesized a series of fibrate analogs that replaced the urea bridge with an amide bridge and modified the substitution on ring A. Compounds from the series exhibited greater allosteric activity than benzafibrate. The most potent derivatives from the series were RSR4 and RSR13, as shown in Table 1 above.
X-ray crystallography studies of bezafibrate complexed with Hb showed that two symmetrically related molecules bind near the top of the xcex1 subunits and point down the central water cavity to the xcex1,xcex2-subunit interfaces making several important interactions with the protein. The high resolution x-ray crystal structure of the RSR13-Hb complex showed that the molecule binds similarly to bezafibrate. The carboxylic acid group of RSR13 forms a water-mediated salt bridge with Arg 141xcex1, the amide oxygen makes a hydrogen bond with Lys 99xcex1 and the gem dimethyl group lies in a hydrophobic pocket lined with residues Pro 95xcex1, Tyr 140xcex1, and Trp 37xcex2.
The present invention was designed to investigate the effect of chirality on the allosteric activity of a series of modifiers and determine their effect on binding mode with Hb. This invention describes the synthesis of several chiral allosteric modifiers of Hb which replaced the gem dimethyl group with alkyl groups, substituted, cycloalkyl groups, and cycloalkyl groups with heteroatoms in the ring. The compounds were based on the ring A templates of RSR13, RSR46, JP7, RSR4 and KDD86. In addition, the structure of JP7 was also modified by adding-substituents to the cyclopentyl ring to give substituted chiral derivatives. Select compounds from the RSR13 and JP7 series were resolved into the enantiomers to determine the effect of the stereocenter on activity. Molecule selection for separation was based on degree of activity and substitution pattern. To formulate SAR, the prepared derivatives were analyzed with Hb solution. In vitro testing with whole blood was also conducted. From studying the binding site of RSR13 and the position of the gem dimethyl group, it was anticipated that one of the enantiomers will bind differently to the hydrophobic pocket and therefore have different effects on the allosteric equilibrium.
All reagent and starting material used in the syntheses were purchased from Aldrich, Fluka, or Sigma and used without purification. All solvents were purchased form Aldrich or Fisher. Silica gel coated plates (0.25 mm thickness) from Analtech, Inc. were used for thin layer chromatography (TLC). Separations were visualized by ultraviolet (UV) lamp or by iodine exposure. Column chromatography was performed on silica gel (Merc, grade 9385, 230-400 mesh). Melting points (mp) were determined on a Thomas-Hoover melting point apparatus and were uncorrected. Proton nuclear magnetic resonance (H NMR) spectra were obtained on a Varian Gemini 300 MHz Spectrophotometer and are reported in parts per million (xcex4 ppm) with tetramethylsilane as the internal standard. Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross, Ga.) and results are within xc2x14% of the theoretical value. All intermediate compounds were analyzed but are not reported. Their purity was determined by TLC and 1H NMR.